Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides.
Endosomal escape and nuclear localization are two barriers to gene delivery that need to be addressed in the design of new nonviral gene delivery vehicles. We have previously synthesized low-toxicity polyethylene glycol (PEG)-based vehicles with endosomal escape functionalities, but it was determined that the transfection efficiency of PEG-based vehicles that escaped the endosome was still limited by poor nuclear localization. Two different nuclear localization signal (NLS) peptides, SV40 and TAT, were coupled to PEG-based vehicles with DNA-binding peptides (DBPs) to determine the effect of NLS peptides on the transfection efficiency of PEG-based gene delivery vehicles. Coupling one SV40 peptide, a classical NLS, or two TAT peptides, a nonclassical NLS, to PEG-DBP vehicles increased the transfection efficiency of PEG-DBP/DNA particles 15-fold and resulted in similar efficiency to that of a common cationic polymer vehicle, polyethylenimine (PEI). The transfection efficiency of both types of PEG-DBP-NLS particles was further increased 7-fold in the presence of chloroquine, suggesting that the transfection efficiency of PEG-DBP-NLS particles is limited by their ability to escape the endosome. To develop particles that could escape the endosome and target the nucleus, a mixture of PEG-DBP-NLS vehicles and PEG-based vehicles with DBPs and endosomal escape peptides were complexed with plasmid DNA to form multifunctional particles that had a transfection efficiency 2-3 times higher than that of PEI. Additionally, the PEG-based vehicles were less toxic and more resistant to nonspecific protein adsorption than PEI, making them an attractive alternative for nonviral gene delivery.
Moore
Nicole M
NM
Department of Environmental, Energy, and Chemical Engineering, Washington University, One Brookings Drive, Saint Louis, MO 63130-4899, USA.
Sheppard
Clayton L
CL
Sakiyama-Elbert
Shelly E
SE
D000818:Animals; D002021:Buffers; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D011992:Endosomes / Q000378:metabolism*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D019913:Nuclear Localization Signals / Q000737:chemistry* / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology; D010710:Phosphates / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011094:Polyethyleneimine / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D002021:Buffers; C526981:INF7 peptide; D019913:Nuclear Localization Signals; D010455:Peptides; D010710:Phosphates; D049452:Green Fluorescent Proteins; D011092:Polyethylene Glycols; D011094:Polyethyleneimine